This invention relates to the field of molecular biology and production of single chain polypeptides.
The immunoglobulin (Ig) superfamily is a group of proteins having as a common denominator an adhesion function or binding function that triggers a subsequent event at a cell surface. The molecules of the Ig superfamily have a diversity of functions, including involvement in immune response and in controlling the behavior of cells in various tissues. See, for example, Williams et al., Ann. Rev. Immunol., 6, 381-405 (1988); Williams, Immunology Today, 8, 298-303 (1987); and Matsunaga, Immunology Today, 6, 260-263 (1985).
The Ig superfamily includes such functionally-important members as the antigen receptors of lymphocytes [e.g., immunoglobulins, T-cell receptor (TCR) molecules, and Class I and Class II molecules of the major histocompatability complex (MHC)]. Lymphocytes are divided broadly into cells that mature in the thymus (T-lymphocytes or T-cells) and cells that mature without passing through the thymus (B lymphocytes or B cells). T-cells can be divided into a number of subtypes such as T-helper cells or cytotoxic T-cells. Helper T-cells respond only to antigen that is on the surface of an antigen presenting cell that also expresses a class II MHC. Cytotoxic T-cells destroy other cells that have been infected by an organism, such as a virus, by recognizing foreign antigen presented in the context of the class I MHC molecule on the surface of the infected cell.
A structural feature that characterizes members of the Ig superfamily is the presence of one or more regions homologous to the basic structural unit of immunoglobulins, the immunoglobulin homology unit. These regions are usually between 10 and 110 amino acid residues in length and are characterized by a series of anti-parallel .beta. sheets that generate a compact sandwich of two .beta. sheets or a single large sheet in the case of MHC proteins. This structure is stabilized by a small series of conserved residues, particularly, two virtually invariant cysteine residues that generate a signature disulfide bond holding the faces of the .beta. sheet sandwich together. The loop sequences connecting the .beta. strands are less significant for the basic homology unit structure and therefore, can accommodate extensive variability.
The Ig superfamily contains distinct classes of immune receptors, molecules that interact directly with an antigen including: (1) antibodies, (2) T-cell receptors, (3) MHC class I, (4) MHC class II; (5) Immunoglobulin Fc receptors (including CD16, CD23, CDw32, etc.) and (6) some cell-surface adhesion molecules (including but not limited to CD4 and CD8 and .beta.1 integrin family-of proteins). Antibodies can be expressed either on the cell surface or humorally; that is secreted into the blood to carry out their functions at a distance from the cell of origin. T-cells synthesize only a surface-bound antigen receptor. There are two classes of receptors encoded within the MHC. Class I MHC molecules are present in varying amounts on virtually all somatic cells. Class II MHC molecules are expressed only on particular antigen-presenting cells such as macrophages and B cells. Class I and class II MHC play a role in presenting antigens in a form which can be recognized by the T-cell receptors. Immunoglobulin Fc receptors are expressed on a variety of hematopoietic cells and function in antibody-dependent T-cell killing and in mast cell degranulation. Adhesion molecules are cell surface proteins involved in regulating the adhesion, migration and overall trafficking of lymphocytes necessary for their immunological function.
The basic structure of an antibody is a tetramer composed of two identical heterodimers each consisting of a light and a heavy chain. The antibody molecule is divided into variable (V) domains that are responsible for binding specificity and constant (C) domains that carry out various effector functions. The V domain is constructed of one V homology unit from both the light and heavy chains, designated V.sub.L and V.sub.H respectively. The variable region can be further subdivided into framework regions (FR) and complementarity determining regions (CDR), also designated as hypervariable regions. The FR maintain the structural integrity of the variable region domain. The CDR are the polypeptide segments within the variable region that mediate binding to the antigen. As the antibody contains two heterodimers, it has two antigen binding sites (divalent).
The T-cell antigen receptor is a heterodimer with one V domain involved in antigen recognition and one C domain that interacts with a membrane-bound protein complex, CD3, presumably involved in signal transduction. See for example Davis, Annu. Rev. Immunol., 3, 537- (1985); Kronenberg et al., Annu. Rev. Immunol., 4, 529- (1986). The majority of T-cells responsible for general antigen recognition use .alpha. and .beta. chains. A small subset of T-cells employ T-cell receptors composed of .gamma. and .delta. chains. As with antibodies, the V domain contains hypervariable regions which are the primary sequences responsible for antigenic interaction. Unlike antibodies, however, these hypervariable regions are not subject to somatic mutation during T cell selection and maturation.
Class I MHC molecules are transmembrane, glycosylated polymorphic polypeptide chains in close, non-covalent association with a .beta.2 microglobulin. The glycosylated polypeptide chain is divided into five distinct regions: three extracellular domains, a transmembrane region, and a cytoplasmic domain; the three extracellular domains designated .alpha.1, .alpha.2 and .alpha.3. Most of the sequence diversity of class I MHC molecules lies in the .alpha.1 and .alpha.2 domains with little sequence diversity in the .alpha.3 domain. The .alpha.1 and .alpha.2 domains interact to form a tertiary structure that is capable of binding a wide array of peptides. In this way, the .alpha.1/.alpha.2 interface is analogous to the V.sub.H /V.sub.L interface of antibodies of the V.alpha./V.beta. interface of the T-cell receptor.
Class II MHC molecules including the I-E, I-A molecules in the mouse, the HLA-DP, DQ and DR molecules in man and equivalent molecules in other mammals) are heterodimeric cell surface proteins composed of .alpha. and .beta. chain polypeptides. With the exception of I-E, HLA-DR and their equivalent genes in other organisms, both the .alpha. and .beta. chains of class II MHC proteins are highly polymorphic. Both chains have two extracellular domains, .alpha.1 and .alpha.2 for the .alpha. chain, and .beta.1 and .beta.2 for the .beta. chain. In each chain, there is also a transmembrane segment and a short intracytoplasmic domain. The .alpha.2 and .beta.2 resemble immunoglobulin constant region domains. The highest degree of sequence variation occurs in the .beta.1 domain with the .alpha.1 and .beta.1 domains forming the recognition site for binding antigen.
By using current techniques in molecular biology, it is possible to clone polypeptide chains. The more complex the molecules, e.g., the multichain immune receptors of the Ig superfamily, the more difficult they are to produce in a single foreign host. Genes coding for each chain of a complex molecule can be cloned and expressed in separate hosts but the aggregation and refolding of the resultant polypeptides into a biologically active entity is difficult to achieve. Multiple chains expressed by multiple genes within the same host have an advantage in aggregating and refolding into native structure but their expression in stoichiometric amounts is difficult to regulate. Thus, neither approach has proven to be efficient.
Since it is the variable regions of light and heavy chain antibodies that interact with an antigen, single chain polypeptides have been created with one V.sub.L and one V.sub.H joined by a peptide linker (U.S. Pat. No. 4,946,778). This single chain fragment (Fv) is univalent and is one of the smallest structures necessary for antigen binding activity having all 6 CDR regions of a V.sub.L -V.sub.H combination. Similarly, the formation of single chain polypeptides containing the variable domains of .alpha..beta. T-cell antigen receptors have also been reported. Novotny et al., Proc. Natl. Acad. Sci. USA, 88, 8646-8650 (1991); Soo Hoo et al., Proc. Natl. Acad. Sci. USA, 89, 4759-4763 (1992). The ability to produce as single chain polypeptides the antigenic or ligand binding portions of antibodies and .alpha..beta. T-cell antigen receptors reduces the difficulties discussed above which are encountered when producing multichain polypeptides.
The use of the functional smaller polypeptides which maintain an antigen recognition site are also advantageous when used for therapeutic purposes in mammals in that the smaller molecules are capable of rapidly localizing in the target tissue, such as single chain antibodies localizing in cancerous tissue or single chain T-cell receptors localizing to the inflammatory sites of patients suffering from such T-cell dependent pathological conditions as acute or chronic graft rejection, graft-vs-host disease, rheumatoid arthritis and other autoimmune diseases. Similarly, soluble single chain MHC molecules (perhaps bound to the disease-propagating antigens) could localize to and block pathologic antigen presentation at sites of T-cell dependent inflammation. However, while being able to penetrate the desired tissue, polypeptides with a molecular weight of less than about 50,000 daltons have the disadvantage of being retained within the glomerulus of the kidney. This is particularly disadvantageous when the polypeptide is bound with a carrier, such as a radioisotope or toxin, as the isotope or toxin will also accumulate in the kidneys.
Single chain polypeptides also suffer from the disadvantage of having only one binding site, thereby reducing their avidity.
It would therefore be advantageous to obtain constructions of the Ig superfamily which retain their antigen or ligand recognition properties, are of sufficient molecular weight to enhance retention at the target site with reduced retention in the kidney, and have a multiplicity of binding sites to enhance the avidity of the polypeptide. In addition, it would be beneficial if the single chain immunoglobulin superfamily proteins could be rendered bispecific to allow for recognition of different epitopes on the target tissue (or lymphocyte) or to allow for antibody-based recruitment of other immune effector functions (i.e., complement proteins, cytotoxic lymphocytes, etc.).